Charged phospholipid compositions and methods for their use

ABSTRACT

The invention provides a pharmaceutical composition comprising a synthetic or naturally occurring charged phospholipid, which is formulated into a dosage form for administration to a subject or which is administered as a food additive. Negatively charged phospholipid composition increase the net negative charge on intravascular lipoproteins, enhance the clearance of cholesterol and regulate the function of lipolytic enzymes, retard prothrombin formation and aid in the clearance of virus and bacterial particles. Negatively charged lipid compositions can therefore be administered to humans and animals for the treatment of hyperlipidemia and blood coagulation disorders and to reduce the levels of virus, bacteria, and endotoxins in the blood stream. Positively charged lipid compositions can be administered to delay lipoprotein clearance from the plasma compartment and give longer duration of activity for drugs which are associated with lipoproteins.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.11/342,867 (titled “Charged Phospholipid Compositions and Methods forTheir Use”), filed Jan. 31, 2006, which is a continuation of U.S.application Ser. No. 10/956,065, filed Oct. 4, 2004), which is acontinuation of U.S. application Ser. No. 10/148,391 (now U.S. Pat. No.6,828,306), which is the U.S. National Phase application of PCTapplication PCT/CA2001/001102 (having an International filing date ofJul. 31, 2001), which claims priority from U.S. Ser. No. 60/221,916(filed Jul. 31, 2000).

FIELD OF THE INVENTION

This invention relates to charged lipid compositions. In one aspect,this invention relates to negatively charged (anionic) lipidcompositions, and the use of these lipid compositions for changinglipoprotein charge in vivo to clear cholesterol and other substancesfrom the blood stream. In another aspect, this invention relates topositively charged (cationic) lipid compositions, and the use of thesecompositions to prolong drug activity.

BACKGROUND OF THE INVENTION

Atherosclerosis leading to coronary vascular disease is a primary causeof mortality in the developed world. Atherosclerotic risk has been shownto be directly related to elevated plasma cholesterol levels. In plasmaabout 70% of cholesterol is esterified to long-chain fatty acids to formcholesteryl esters and these cholesteryl esters are bound to plasmalipoproteins. The lipoproteins involved in the transport of cholesteroland cholesteryl esters include low density lipoprotein (LDL), highdensity lipoprotein (HDL), and very-low density lipoprotein (VLDL).

While high levels of cholesterol associated with LDL have been linked toatherosclerotic risk (Schaefer et al., 1995), high HDL cholesterollevels may be protective against the development of heart disease(Miller at al., 1977). As a result there has been significant effort todevelop therapies which effectively reduce the level of LDL cholesteroland raise the level of HDL cholesterol within an animal. HDL may play ananti-atherogenic role by promoting the clearance of cholesterol from thebody (Eisenberg, 1984). Furthermore, Schwartz at al. (1978) disclosethat cholesterol in HDL is specifically targeted for excretion from thebody by the liver in the form of bile. However, current therapiesdirected to reduce the level of LDL cholesterol and raise the level ofHDL cholesterol have not met with success.

The factors that regulate cholesterol flux to the liver are poorlyunderstood but may involve two distinct systems; a cellular sterolregulatory system and an intravascular transport system. Excessextrahepatic cholesterol may be transported in HDL particles to theliver for excretion (Glomset, 1968). HDL has also been shown to be ableto adsorb cholesterol and cholesteryl esters (CE) from cell membranes(Phillips at al., 1998). In addition, a second sterol transport pathwaymay include transfer of cholesterol from HDL to the rapidly turning overVLDL lipoprotein pool, followed by clearance of cholesterol by the liver(Tall, 1998).

The mechanism of intravascular sterol transport is also poorlyunderstood, but may involve the concerted action of multiple proteinsand enzymes. Two enzymes thought be involved in intravascular steroltransport are lecithin:cholesterol acyltransferase (LCAT) andcholesterol ester transfer protein (CETP). At present it is thought thatLCAT may form a concentration gradient to move sterol into and throughthe blood plasma compartment by promoting the conversion of freecholesterol (FC) to cholesterol esters (CE) on HDL particles (Jonas,1987). CETP may then promote this lipid flux by moving the newly formedCE from HDL to an apoB containing lipoprotein pool (Lagrost, 1997).

All lipoprotein classes exhibit a net negative charge, due to both theapolipoprotein composition and its content of bound charged lipids(Davidson et al., 1994). However, individual bound lipids forming partof a lipoprotein can contribute either a net positive or a negativecharge, or no charge at all, to the lipoprotein. Some phospholipids,when unbound, are negatively charged, some are positively charged, andsome are electrically neutral. Examples of negatively chargedphospholipids are phosphatidylinositol, phosphatidylserine,phosphatidylglycerol and phosphatidic acid. An example of anelectrically neutral phospholipid is phosphatidylcholine. An example ofa positively charged (cationic) phospholipid is dioleoyltrimethylammonium propane.

Williams U.S. Pat. No. 6,079,416 teaches administration of largeliposomes containing phospholipids substantially free of sterols totreat hypercholesterolemia. Parker et al U.S. Pat. No. 5,614,507 teachesthe injection of a bolus of phospholipid, with or without anotherelectrically neutral lipid, to treat endotoxemia. However, thephospholipid compositions disclosed by Williams and Parker et al. arenot electrically charged, so they would not act to change the electriccharge of lipoproteins within the bloodstream. Instead, they act as asimple absorbent to pick up and clear cholesterol or endotoxin from theblood.

U.S. Pat. No. 5,652,339 (Learch et al) and U.S. Pat. No. 5,128,318(Levine at al) disclose the preparation of reconstituted high densitylipoprotein (rHDL) particles, and suggest that rHDL particles may beused for drug administration and for treating diseases connected tolipids and lipodal substances. These rHDL particles said to be useableboth in vivo or in vitro for removing lipid soluble materials (e.g.cholesterol, endotoxins) from cells or body fluids and aid in thetreatment of hyperlipidemia and coronary atherosclerosis. Although alllipoprotein classes exhibit a net negative charge, as discussed above,these two patents teach nothing to increase the charge from that presentin normal HDL.

Phosphatidylinositol (PI) is a negatively charged phospholipid found inall classes of lipoproteins and accounts for approximately 4% of thetotal phospholipid (PL) in HDL (Davidson et al., 1994). Incubation of PIwith plasma or with isolated HDL, LDL or VLDL in vitro has shown thatall of these lipoproteins can spontaneously absorb PI. However, littleis known of what affects, or regulates, the amount of PI in differentlipoprotein classes.

There is a need for novel compositions capable of enhancing hepaticclearance of lipoprotein particles thereby lowering cholesterol andtissue cholesterol, endotoxins and other lipid-soluble compounds such assome viruses and bacteria that associate with lipoprotein particles invivo. There is also a need for methods to make use of such compositions.In particular, there is a need for such compositions, and methods fortheir use, which will preferentially lower the cholesterol associatedwith LDL. There is also a need for compositions, and methods for theiruse, which will retain drugs which associate with lipoproteins in thebloodstream, to increase the duration of the efficacy of such drugs.

SUMMARY OF THE INVENTION

The present invention provides negatively charged (anionic) lipidcompositions that, when administered to an animal or subject, result inan increase in the in vivo lipoprotein negative electric charge.Associated with the increase in in vivo lipoprotein negative charge isan enhanced hepatic clearance of lipoprotein particles. The clearance oflipoprotein particles can be used for the clearance of cholesterol andhas significant anti-atherogenic consequences. It can also be used toremove bacteria, endotoxin and viruses which associate with lipoproteinsfrom the bloodstream, and for the treatment of lipid-associateddiseases.

The invention also provides positively charged (cationic) lipidcompositions which, when administered to an animal or subject, result ina decrease of the in vivo lipoprotein negative electric charge.Associated with the decrease in in vivo lipoprotein negative charge is aslowing of hepatic clearance of lipoprotein particles. This effect canbe used to delay clearance of drugs which are associated withlipoproteins, thereby prolonging the efficacy of such drugs.

The present invention also comprises a pharmaceutical compositioncomprising a synthetic or naturally occurring negatively charged(anionic) phospholipid formulated into a dosage form for administrationto a subject. If desired, the composition may comprise an admixture oftwo or more negatively charged phospholipids. The pharmaceuticalcomposition is capable of mediating the level of lipid-associatedcompounds within an animal or subject. The invention also comprises theuse of a negatively charged phospholipid composition as defined abovefor the production of a medicament useable to enhance clearance ofcholesterol from the blood stream and cause the reduction in blood LDLand VLDL cholesterol levels.

The pharmaceutical compositions as discussed above can be provided indosage forms comprising unilamellar vesicles multilamellar vesicles,multilamellar sheets, dispersions, micellar solutions, emulsions,microemulsions, pure lipid mixtures or any combination of thesestructures.

The present invention also relates to compositions as discussed above,wherein the dosage form is administered orally or as an injectionintranasally or transdermally. In addition the composition may furthercomprise at least one pharmaceutically acceptable carrier.

The present invention also comprises an orally-administered foodadditive which comprises a charged phospholipid. When the chargedphospholipid is a negatively charged phospholipid, the food additive iscapable, when ingested by a subject, of reducing the amount ofcholesterol in the subject's bloodstream. A particularly preferred foodadditive comprises phosphatidylinositol.

The present invention also relates to preferred compositions of thosediscussed above, wherein the charged phospholipid is a negativelycharged phospholipid selected from phosphatidylinositol,phosphatidylserine, phosphatidylglycerol and/or phosphatidic acid ormixtures thereof, and particularly preferred compositions wherein thenegatively charged phospholipid is phosphatidylinositol.

The invention also relates to a process for enhancing clearance oflipoprotein particles in vivo by administration of at least onenegatively charged phospholipid. By increasing lipoprotein clearance,lipid-soluble compounds that associate with lipoprotein particles arealso effectively removed. For example, as described herein,administration of charged phospholipid results in sterol mobilizationinto bile and excretion in faeces. Removal can therefore be accomplishedof cholesterol, endotoxins, or lipoprotein-associated bacteria or virusparticles. Without wishing to be bound by theory, it is thought that theadministration of a pharmaceutical charged lipid composition to ananimal or subject lowers serum levels of total cholesterol in the animalor subject, and inhibits the conversion of cholesterol to cholesterylesters. Lipoprotein charge thus plays a role in regulating serum levelsof lipoprotein-associated compounds, for example but not limited to freecholesterol and the like.

Also according to the invention, there is provided a method of loweringthe level of cholesterol associated with LDL within a subject, themethod comprising administering to the subject an effective amount of acomposition comprising an anionic phospholipid composition.

Another aspect of the present invention pertains to the enhancedmobilization of cellular sterol, and the promotion of rapid clearance ofboth FC and CE from the plasma compartment, following the administrationof a negatively charged phospholipid, (in a particularly preferredembodiment, PI) to a subject. Without wishing to be bound by theory, itis proposed that lipoprotein charge can affect cholesterol transport andthat this process can be selectively manipulated by manipulatinglipoprotein charge.

Other aspects of the invention provide methods of removing endotoxins,bacteria and virus particles, and treating lipid-associated diseaseswithin an animal, including hyperlipidemia and atherosclerosis, byadministering a negatively charged phospholipid to the animal orsubject.

Another aspect of the invention provides a method for slowing theremoval of lipoproteins, and drugs which associate with lipoproteins,from the bloodstream of an animal or subject by administering apositively charged phospholipid to such animal or subject. The inventionalso comprises compositions of positively charged phospholipids usefulfor such methods.

BRIEF DESCRIPTION OF THE DRAWINGS

These and other features of the invention will become more apparent fromthe following description in which reference is made to the appendeddrawings wherein:

FIG. 1 depicts graphically the change in the negative surface charge ofHDL, LDL and VLDL proteins following administration ofphosphatidylinositol (PI) or phosphatidylcholine (PC) vesicles torabbit.

FIG. 2 shows a graphical representation of the effect of PI onlipoprotein cholesterol levels in plasma from a fasted normolipidemicsubject

FIG. 3 shows the effect of injection of PC or PI into rabbits on plasmacholesterol esterification.

FIG. 4 shows the effect of incubations with PC or PI on the lipoproteinlevels of FC and CE in human blood.

FIG. 5 shows the time clearance of [³H]-FC from the blood stream of arabbit following administration of PI and PC vesicles containing[³H]-FC.

FIG. 6 shows the effect of PI on biliary FC output. Rabbits wereinjected with PI or PC vesicles containing [³H]-FC and then sacrificedat 30 min.

FIG. 7 shows the effect of PI or PC injections into rabbits on faecalcholesterol excretion.

FIG. 8 shows the effect of PI injections into hypercholesterolemicrabbits on HDL, LDL and VLDL cholesterol levels.

FIG. 9 shows the effect of the oral administration of PS on HDL chargein a human subject.

FIG. 10 shows the effect of lipoprotein charge on lipolysis by hepatictriglyceride lipase.

FIG. 11 shows the effect of charged lipids on plasma clotting time.

DESCRIPTION OF PREFERRED EMBODIMENTS

Lipoprotein charge is affected by its content of charged molecules,predominantly charged lipids such as PI and non-esterified free fattyacids (NEFA). PI is an anionic lipid found in all classes oflipoproteins and accounts for approximately 4% of the total phospholipidin HDL (Davidson et al. 1994)

The present invention provides for a pharmaceutical compositioncomprising a charged phospholipid, either natural, analog, or acombination thereof, and in some cases, a suitable carrier. A suitablecarrier may include, but is not limited to, phosphate buffered saline,sodium cholate, uncharged synthetic or natural ampliphatic lipid, HDLparticles, or any protein capable of binding one or more chargedphospholipids. The carrier can be one which permits delayed or timedrelease of the phospholipid, as is known in the carrier art.

The negatively charged (anionic) phospholipids of the invention includeany negatively charged phospholipid that increases the negative chargeof lipoprotein in vivo. Preferred negatively charged phospholipids ofthe present invention are phosphatidylinositol (PI), phosphatidylserine(PS), phosphatidylglycerol (PG), phosphatidic acid (PA), or a mixture ofany of these phospholipids. A particularly preferred phospholipid isphosphatidylinositol (PI).

The positively charged (cationic) phospholipids of the invention includeany positively charged phospholipid that decreases the negative chargeof lipoprotein in vivo. A preferred positively charged phospholipids isdioleoyl trimethylammonium propane (DOTAP).

The negatively charged phospholipid composition can be administered overa range of dosages that enhance clearance of cholesterol or othersubstances to be removed. When used in a pharmaceutical composition,administration in an amount from about 5 micromole to about 100micromole per kg body weight of an animal or human subject is suitable.Preferably, the level of charged phospholipid administered to an animalor human subject is from about 5 micromole to about 20 micromole per kgbody weight administered either in a single dose, or over a short period(e.g. one hour). For example, which is not to be considered limiting,administration of PI in an amount from about 5 micromole to about 100micromole per kg body weight of an animal or human subject enhancescholesterol mobilization as described herein.

When administration occurs by injection, the amount administered shouldpreferably be enough to cause an increase in the negative surfacepotential on the surface of the HDL fraction of the blood of at leastabout 20% but preferably not more than 80% (i.e. from a normal value ofabout −11 to −12.2 mV to a value of about −18 to −22 mV) for a transientperiod. Generally, the period lasts for at least one hour, and commenceswithin a time of one minute to two hours after delivery to the body ofthe animal or human subject, although individual reactions may vary. Thenegative surface potential can conveniently be measured by removal ofblood samples from the animal or human subject, and observing themigration of the lipoproteins in an agarose gel, in the manner known inthe art. It is found that oral administration causes a less markedchange in charge of the HDL fraction than administration by injection,(for example, not to be considered limiting, from about −10.9 my toabout 12 mV), but that the transient period lasts longer with oraladministration.

Subsequent doses, or continuous administration at a low level for asuitable period of time, can be given to maintain the increased negativesurface potential for a longer period than single dose administrationwould give.

In one embodiment, the charged phospholipid composition can be used forthe production of a medicament. The medicament containing a negativelycharged phospholipid can be used to enhance clearance of cholesterol, orto remove endotoxin or virus particles. The medicament containing thepositively charged phospholipid can be used to slow lipoproteinclearance from the blood stream, and thereby slow removal from thebloodstream of a drug that associates with lipoproteins. The medicamentcan optionally also contain a drug that associates with lipoproteins,for example a drug associated with HDL or rHDL particles.

The medicament can be delivered by injection, or transdermally (as by apatch or a subcutaneous injection) or orally. The carrier is chosen, asknown to one skilled in the art, to be compatible with the type ofadministration. For some types of administration, no carrier isnecessary.

In another embodiment, the charged phospholipid composition can beincluded in a food. The food can than be administered to the subject,enhancing that subject's clearance of cholesterol. The phospholipidcompositions can for example be dried and admixed into solid foods, orthey can be mixed as an emulsion into liquid foods which will notneutralize them (for example, milk), or they can be sprayed onto solidfoods (such as for example cereals) and allowed to dry before the foodsare administered.

To demonstrate the role lipoprotein charge plays in cholesterolmetabolism in vivo, intravenous injection of an uncharged phospholipid(phosphatidylcholine, PC) or a charged, anionic phospholipid{phosphatidylinositol, (PI) or phosphatidylserine (PS)}—into a fastedrabbit was examined. Similar effects occur with other test animals. PCinjection has a negligible effect on lipoprotein charge and composition,similar to that observed in a saline injected animal. However, PIinjection causes a significant increase in the net negative surfacecharge of all lipoproteins (see FIG. 1, discussed further below). Thischange in net negative surface charge is observed rapidly, for example,but not limited to after about 10 min, and is followed by a gradualreturn to regular levels by about 24 h, however, these time periods mayvary depending upon the animal being treated. Alterations in the netcharge of lipoproteins has also been observed when plasma is incubatedin the presence of PI or PS. PI and PS show similar results.

In the studies conducted as described herein, no major changes in thelevels of, or the composition of, HDL or LDL are evident over a 24 hturnover period. Co-injection of [³H]-FC revealed an increase in therate of clearance of labelled cholesterol from the PI injected rabbitplasma (see FIG. 5, discussed further below). In addition, the rate ofcholesterol esterification by lecithin:cholesterol acyltransferase wasalmost completely inhibited in the PI animals (See FIG. 3, discussedfurther below).

By the term “charged phospholipid” is meant a natural or syntheticglycerophospholipid which is electrically charged at neutral pH. A“negatively charged phospholipid” (also known as an “anionicphospholipid”) has a negative charge at neutral pH. A “positivelycharged phospholipid” (also known as a “cationic phospholipid”) has apositive charge at neutral pH.

Persons skilled in the art will understand that glycerophospholipidscomprise a glycerol backbone wherein one of the hydroxyls is linked to apolar phosphate-containing group and the other two hydroxyls are linkedto hydrophobic groups. Also evident to someone skilled in the art isthat glyceride nomenclature is often defined in terms of astereospecific numbering (sn) system, but that other stereochemicalconventions such as D/L and R/S may be used, with most naturalglycerophospholipids having the R or D configuration. The presentinvention contemplates charged phospholipids having either R or S (orequivalently D or L) configurations.

Most natural glycerophospholipids have hydrophobic groups attached tothe sn-1 and sn-2 positions of the glycerol backbone, while a phosphateis usually attached at the sn-3 position. The hydrophobic groups usuallycomprise long chain hydrocarbons that are linked through ester or etherlinkages. Thus, the present invention fully contemplates chargedphospholipids comprising hydrophobic groups linked through ester orether linkages. The hydrophobic groups may be any known in the art, forexample but not wishing to be limiting, the hydrophobic groups maycomprise saturated fatty acids such as lauric, myristic, palmitic,stearic, arachidic acid, or unsaturated fatty acids such as, but notlimited to palmitoleic, oleic, linoleic, arachidonic acid, or anycombination thereof.

The phosphate group of a glycerophospholipid is usually attached to theglycerol moiety at the sn-3 position and this phosphate group is linkedto a head-group moiety. In phosphatidylinositol, the headgroup isinositol, while in phosphatidylserine, the headgroup is serine. Thecharged phospholipids of the present invention can have a phosphategroup esterified to any hydroxyl of glycerol, with the remainingglyceryl hydroxyls esterified or attached via an ether bond to ahydrophobic group as defined above. In addition, the headgroup mayoptionally be substituted.

By the term “pharmaceutical composition” or “charged phospholipidcomposition” it is meant a composition comprising a chargedphospholipid, as discussed above, that is formulated into an appropriatedosage form in the presence of a suitable carrier for administering to asubject. The carrier may consist of a buffer, for example but notlimited to phosphate buffered saline, or a protein capable ofassociating with an charged phospholipid as described herein. Forexample, without limitation, the charged phospholipid can be formulatedinto structures such as unilamellar vesicles, multilamellar vesicles,multilamellar sheets, dispersions, micellar solutions, emulsions,microemulsions, or any combination thereof in the presence of phosphatebuffered saline and the like as would be known to one of skill in theart, HDL particles, or any protein capable of associating with orbinding charged phospholipid.

The formulations described above represent aqueous formulations.However, the charged phospholipid can be formulated as a solid, such as,without limitation, a powder that may form structures such as one ormore of those discussed above when added to an aqueous solution. Also,the pharmaceutical composition may optionally include the addition ofone or more pharmaceutically acceptable excipients as would be known tosomeone of skill in the art. For example, but not meaning to belimiting, the pharmaceutical composition may comprise other lipids tofacilitate the formation of vesicular structures in solution.Alternatively pharmaceutical acceptable excipients such as but notlimited to, hydrophilic phase components, lipophilic phase componentsand surfactants may be optionally included if the charged phospholipidis to be delivered in the form of an emulsion or microemulsion.Pharmaceutically acceptable excipients may also include, but are notlimited to, solvents, buffers, antioxidants, and stabilizers as is knownin the art.

The pharmaceutical composition of the present invention may beadministered to a subject by any method known in the art. For example,and without limitation, the pharmaceutical composition can beadministered orally or the pharmaceutical composition may be injectedsuch as but not limited to intravenous injection, intramuscularinjection, or intraperitoneal injection. However someone of skill in theart will understand that certain dosage forms may be more suitable tospecific modes of administration. For example, if the pharmaceuticalcomposition is to be administered intravenously as a vesicular solution,it is preferable for the charged phospholipid composition to beadministered as unilamellar vesicles of cross-section sufficiently smallso that they will not get caught in the microvasculature of a subject.

When the charged phospholipids of the present invention are administeredas a food additive, they can be administered as part of any solid orliquid food with which they do not react or complex in such a way as tolose their charge. It is particularly preferred to administer thecharged phospholipid or a mixture of charged phospholipids having thesame charge in a dry state on or commingled in conventional cerealproducts, although other foods can also be used. In case of either apharmaceutical or a food product, sufficient is administered to resultin a change, for a period of at least 10 minutes, of the in vivo chargeon the blood lipoproteins by at least 20%. The negatively chargedphospholipids increase the negative charge of the lipoproteins, whilethe positively charged phospholipids decrease it.

The invention will be further illustrated in the following examples.However, it is to be understood that these examples are for illustrativepurposes only, and should not be used to limit the scope of the presentinvention in any manner.

Procedures Used in Carrying Out the Examples. a): Preparation ofPhospholipid Vesicles

1-palmitoyl-2-oleoyl-phosphatidylcholine (PC) and 1-palmitoyl-2-oleoylphosphatidylinositol (PI) vesicles are prepared by drying to completion40 μmol of each lipid into a 12×75 mm culture tube under N₂. The lipidsare solubilized in 3 mL of sterile saline comprising 150 mM sodiumchloride (pH 7.2) by sonication for 1 minute at constant duty cycle. Thevesicles are incubated at 37° C. for 10 minutes and then sonicated at ahigh output for 4 minutes in 10° C. water bath under N₂. In order todetermine the rate of clearance of cholesterol from the test animals(rabbits), a radioactive tracer is added to the vesicle preparationsprior to injection into the rabbit. 200 μL of 1 μCi/μmL [³H]-FC is driedinto a 12×75 mm culture tube with 40 μmol of PI or POPC. 3 ml of PBS isadded to the dried lipids and the mixture is sonicated as describedabove. This tracer/vesicle preparation is injected and then 5 min later,the PI or PC vesicles were injected and blood is sampled as describedbelow.

(b): Lipid Injection into Rabbits

Male New Zealand white rabbits (3.5-4.0 kg) are fasted for 12 hoursprior to injection and remain fasted until after data for the final timepoint is taken. Rabbits have free access to water during this time. Acatheter is inserted into the marginal ear vein and blood samples arecollected into tubes containing 7.5% (K₃) EDTA solution at the desiredtime points. A pre-injection blood sample is taken and the vesiclesolution of either PI (n=4), or PC (n=2) or saline (n=1) is injectedinto the marginal ear vein. A sample of blood is removed at 10, 30 min,1, 3, 6 and 24 h after the injection. All blood samples are placed onice and then centrifuged at 3000 rpm for 15 min at 4° C. to separate theplasma. In order to ensure that lecithin:cholesterol acyl transferase(LCAT) is inhibited in the stored plasma, iodoacetamide (150 mM) isadded to plasma samples as taught by Guerin et al. (1994).

Comparative studies are carried out in which the tracer is combined with1 mg of PC and 3 mL of PBS instead of with PI and vesicles were preparedas described above. This is done to verify that PI is not affecting theincorporation/clearance of the tracer. The tracer incorporation andclearance was found not to be affected by the PI.

(c): Characterisation of Lipoproteins

Lipoprotein fractions are isolated by sequential ultracentrifugation(VLDL+IDL, d<1.019 g/mL; LDL, d=1.019-1.063 g/mL; and HDL, d=1.063-1.21g/mL) as known to one of skill in the art, and lipoprotein lipidcomposition (total cholesterol, free cholesterol (FC), and triglyceride(TG) concentrations) are determined enzymatically using kits from RocheDiagnostic (Laval, PQ). An aliquot of each lipoprotein isolated isdialysed into PBS and its surface charge characteristics were determinedby electrophoresis on pre-cast 0.5% agarose gels using the procedure ofSparks and Phillips (1992).

(d): Measurement of Cholesterol Esterification

The effect of PI on LCAT activity is examined using plasma samples thatare not treated with iodoacetamide. 400 μL aliquots of plasma for eachtime point are incubated for 30 min with 10 μCi of [³H]-FC on filterpaper discs (Dobiasova at al., 1991) at 37° C. and the reaction isterminated with the addition of 2 mL ethanol. Reaction products areextracted in hexane and the amount of [³H] associated with CE and FC isdetermined by thin layer chromatography.

(e): Stimulation of Biliary PC Output and Sterol Excretion

Rabbits are injected with 40 μmol PI or PC vesicles containing 400 μCi[³H]-FC. Animals are sacrificed at 30 min, and bile is aspirated fromthe gall bladder. In addition the livers are harvested, homogenized andtissue radioactivity determined. Sterol excretion in the faeces ismeasured similarly except that faecal cholesterol levels are measuredover a time period of 96 hours.

In Examples 1-8 and 11-12, the effect of a negatively chargedphospholipid (PI or PS) is contrasted with the effect of a control. Thecontrol is an uncharged phospholipid (PC).

Example 1 The Effects of Lipid Vesicles on Lipoprotein Surface Charge

Referring now to FIG. 1, there is illustrated the effect of injection ofPI and PC vesicles on the estimated surface potential of HDL, LDL, andVLDL proteins in vivo. Injection of PC or saline (not shown) did notsignificantly affect migration of HDL, LDL and VLDL proteins into anagarose gel whereas injection of PI is associated with increasedmigration of all lipoprotein fractions into the gel. Without wishing tobe bound by theory, the results indicate that the increased migration ofthe lipoprotein fractions following PI injection is attributable to anincreased negative surface charge on the lipoproteins, possibly as aresult of increased binding of the PI charged phospholipid species. PC,which is a neutral phospholipid, may also bind to the lipoproteins.However since PC is a neutral phospholipid, no significant change in themigration of the lipoproteins is observed. The increased migratoryproperty of lipoproteins in a gel peaks following injection of PI andreturns to normal by about 24 hours. This indicates that the increasednegative surface charge imparted to HDL, LDL and VLDL lipoproteins is aresult of charged phospholipid binding following PI injection, and thatthis negative surface charge is transient and reversible. Based on themigration patterns of the lipoproteins after removal from the subject inan agarose gel, it is estimated that the HDL fraction exhibits abackground surface potential of about −12.2 mV, prior to the PIinjection and reaches a peak negative charge of about −18.0 my about 10minutes after PI injection. Similarly, the VLDL fraction is estimated tohave an initial surface potential of about −8.8 mV, which increases toabout −11.2 mV after 10 minutes. LDL is estimated to have an initialsurface potential of about −3.9 mV, and peaks about 60 min after the PIinjection at about −8.0 mV.

This change in surface charge in the phospholipid compositions asdescribed herein, alters the metabolism of lipids and lipid-solublecompounds, for example cholesterol, in such a manner that theselipid-soluble compounds are lowered in a subject as described forcholesterol, below.

Example 2 The Effects of Negatively Charged Phospholipid on LipidTransfer in Plasma

Referring now to FIG. 2, there is graphically depicted the effect ofincubations with PI on the amount of cholesterol associated with HDL(high density lipoprotein), LDL (low density lipoprotein) and VLDL (verylow density lipoproteins) following isolation of lipoproteins fromplasma of fasted normolipidemic subjects. The addition of PI to bloodplasma of a subject is found to be associated with a reduction ofcholesterol in LDL and VLDL and increase in HDL cholesterol levels.Without wishing to be bound by theory, the results indicate that PIstimulates the transfer of CE and FC from LDL and VLDL to HDL. Thisappears due to the electrostatic effects that increased lipoprotein PIconcentrations have on cholesteryl ester transfer protein. Previous workhas shown that this protein controls interlipoprotein cholesteroltransfers and is affected by lipoprotein charge (Lagrost 1997).

Example 3 Inhibition of Cholesterol Esterification by Negatively ChargedPhospholipid

Referring now to FIG. 3, the graph shows the effect of PC or PIinjection within an animal on the rate of cholesterol esterification byLCAT at various times following injection of PI or PC in vivo. Noappreciable change in LCAT activity is observed after injection of PCvesicles. However, endogenous LCAT activity is reduced to about 18% thatof normal (PC injected control) after about 10 min post injection of PI.Also, PI injection reduces the fractional rate of cholesterolesterification from about 45% to about 8%, of initial values, per hour.After a period of time following PI vesicle injection, as shown in FIG.3, LCAT activity returns to about 75% of normal. Without wishing to bebound by theory, these results indicate that PI may inhibit CEproduction by lecithin:cholesterol acyl transferase (LCAT).

To investigate the effect of PI on the production and transport of CE inthe plasma, the amount of radioactive CE was measured at each time point(data not shown). The levels of [³H]-CE in the PI and PC injectedrabbits were significantly different. Most notably, at the initial timepoints, there was 85% less [³H]-CE in the PI injected rabbit, than inthe control. This indicated that the production of CE may be impaired inthe PI rabbits, and therefore the endogenous activity of LCAT in plasmawas measured (FIG. 3). The data shows that enrichment of plasmalipoproteins with PI almost completely inhibits cholesterylesterification by LCAT. Without wishing to be bound by theory, theresults indicate that the reduced levels of [³H]-CE in PI injectedanimals were primarily due to a decreased production by LCAT. Sinceplasma CE levels actually rise in the presence of PI (and LCATinhibition), the data indicate that LCAT is not solely responsible forthe amount of CE in plasma lipoproteins. Rather it appears that PI maymodify intravascular cholesterol levels by mobilizing a cellularcholesterol pool.

Example 4 Effect of Negatively Charged Phospholipids on Lipoprotein-RedBlood Cell Interactions

Referring now to FIG. 4, there is graphically depicted the effect of PIor PC vesicles on lipoprotein CE and FC. Incubation of whole blood withPC vesicles results in a 60% increase in the CE content of thelipoproteins, while FC content is reduced by about 10%. In contrast, PIvesicle incubation with whole blood indicates that lipoprotein CE isreduced by about 40% while PC levels increase by about 40%. Negativelycharged phospholipids appeared to promote a reciprocal exchange of CEfor FC between red blood cells and the lipoprotein particles. However,since CE did not accumulate in the red blood cells, the experiment showsthat PI stimulates a red blood cell associated CE hydrolase that canhydrolyse lipoprotein CE. It has previously been taught that red bloodcells cannot store CE and therefore utilize a membrane-bound CEhydrolase to maintain membrane FC levels. Without wishing to be bound bytheory, the results therefore indicate that PI stimulates thedegradation of lipoprotein CE.

Example 5 Effect of PI on the Clearance of [³H]-FC from the Blood of aRabbit

Referring now to FIG. 5, there is shown the clearance of [³H]-FC atvarious times after rabbits are injected with [³H]-FC and either PI orPC vesicles. FIG. 5 shows that [³H]-FC in plasma of PI injected rabbitsfalls to about 15% of the initial dose after about 1 h. In comparison,rabbits injected with PC contain approximately 90% of the injectedradioactivity after 1 h. Further, the PC injected rabbit requires longerthan about 6 h to clear the [³H]-FC to a comparable baseline level, whencompared with an animal injected with PI, under identical conditions.The half-life of [³H]-FC in the PI injected rabbits is determined to beabout 0.3 h and is about 30-fold shorter than that for the PC injectedrabbits (about 8.4 h).

To verify that [³H]-FC was not affected by co-injection of [³H]-FC withdifferent lipids, [³H]-FC was combined with a small amount of PC,injected into a rabbit and then PI vesicles were injected 5 min later.These experiments showed a similar rapid clearance of [³H]-FC as wasseen with the co-injection of PI and [³H]-FC.

Example 6 Effect of Negatively Charged Phospholipid on Biliary FC Output

Referring now to FIG. 6, there is shown the effect of PI on biliary FCoutput after rabbits are injected with PI or PC vesicles containing[³H]-FC. In comparison to PC, PI injection is associated with areduction of [³H]-FC in plasma, and an elevation of [³H]-FC in theliver. Without wishing to be bound by theory, PI may enhance theclearance of cholesterol from plasma and promote its uptake andaccumulation in the liver. In addition, PI injection appears to beassociated with an increase output of cholesterol into bile. About20-fold enhanced output of cholesterol into bile is observed (FIG. 6).These results suggest that PI is capable of lowering plasma cholesterollevels by stimulating hepatic uptake and excretion of free cholesterolin bile.

Example 7 Effect of Negatively Charged Phospholipid on Sterol Excretion

Referring now to FIG. 7, there is graphically depicted the effect of PIinjection associated with sterol excretion. FIG. 7 suggests that PIinjection enhances faecal cholesterol output. PC injection into a rabbithad no effect on cholesterol excretion, while PI injection enhancedfaecal cholesterol output by about 300% at 2 days post injection. Theincrease in cholesterol output returned to normal by about day 4. Theseresults show that PI can stimulate sterol excretion.

Example 8 Effect of Negatively Charged Phospholipids on the Treatment ofHypercholesterolemia

Referring now to FIG. 8, there is graphically depicted the effect of PIinjection on lipoprotein cholesterol levels in hypercholesterolemicrabbits. Rabbits were fed a diet enriched in cholesterol for a period of4 weeks to increase the levels of cholesterol in the LDL and VLDLparticles in the bloodstream. One half of the study rabbits were theninjected with 36 mg of PI 2× daily for 7 days. On day 7, plasma sampleswere drawn and lipoproteins were fractionated by size exclusionchromatography on two Superose 6 columns. Shown are gel filtrationprofiles for the different rabbit plasmas, which illustrate the levelsof cholesterol in the various lipoprotein fractions. The untreatedhypercholesterolemic rabbit plasma sample is shown to have high levelsof LDL and VLDL cholesterol. PI injections caused a greater than 7-foldreduction in VLDL cholesterol and about a 2.5-fold reduction in LDLcholesterol levels over the 7 day period. In contrast, PI slightlyincreased the amount of cholesterol in the HDL particles. PI thereforecan be used to treat hypercholesterolemia and reduce plasma LDL and VLDLcholesterol levels.

Example 9 Effect of Orally Administered Negatively Charged Lipids onLipoprotein Charge

Referring now to FIG. 9, there is graphically depicted the results oforal administration of 300 mg of PS to a human subject. Plasma sampleswere drawn before, and over a 24 hour period after, administration ofPS. HDL charge was determined electrokinetically after electrophoresisof the plasma samples on 0.5% agarose. HDL charge progressivelyincreased from −10.9 mV to over −12 mV by 6 hours, and then fell to−11.4 mV by 24 hours post injection. Similar results have been observedwhen PI was dried onto rabbit chow and then fed to rabbits (data notshown). Thus, orally administered negatively charged lipids is absorbedintestinally and alters lipoprotein charge in the blood stream.

Example 10 Effect of Lipoprotein Charge on Lipoprotein Lipase andHepatic Lipase

Referring now to FIG. 10, there is shown the effect of lipoproteincharge on hepatic lipase. High density lipoprotein particles wereenriched to contain various amounts of anionic or cationic lipids andthen characterized as substrates for pure human hepatic lipase.Decreasing the net negative charge on the lipoprotein was associatedwith a reduction in the lipolytic activity of hepatic lipase.Lipoprotein charge can also be used to regulate lipoprotein lipase.Enriching plasma with PI was shown to stimulate lipid hydrolysis bylipoprotein lipase, while addition of the cationic lipid, DOTAP, had theopposite effect and inhibited lipid hydrolysis (not shown). Lipidhydrolysis by hepatic or lipoprotein lipase can therefore be affected byalterations in the charge properties of plasma lipoproteins.

Example 11 Effect of Charged Lipids on Plasma Clotting Time

Referring now to FIG. 11, there is shown the effect of charged lipid onblood coagulation. Human plasma was incubated with various amounts ofuncharged vesicles, phosphatidylcholine (PC) or anionic vesicles,phosphatidylinositol (PI), for 24 h at 4° C. Prothrombin time was thenmeasured with the use of a commercially available kit. The results areshown in FIG. 11. Increasing amounts of PC has no significant effect onplasma clotting time, while PI caused an inhibition to the formation ofprothrombin.

Example 12 Effect of Charged Lipid on the Clearance of Cytomegalovirusfrom the Plasma of a Rabbit

Rabbits were injected with 8×10¹⁰ cytomegalovirus (CMV) particles andthen with 36 mg of either uncharged vesicles, phosphatidylcholine (PC),or anionic vesicles, phosphatidylinositol (PI). Blood samples were takenat specific intervals and the amount of virus in the blood samples wasdetermined by immunometric analysis. The results were demonstrated in aCMV p28 western blot of a blood sample taken at 10 minutes postinjection (not reproduced in this document). PI caused a 1.7-foldincreased rate of clearance of the CMV injectate relative to thatobserved with PC.

Example 13 Effect of Anionic and Cationic Lipids on Lipoprotein Chargein Human Plasma

Different concentrations as shown below of negatively chargedphospholipid (PI) or positively charged phospholipid (DOTAP) were addedto human plasma and the surface charge on the lipoproteins wasdetermined. The human plasma was incubated with PI or dioleoyltrimethylammonium-propane (DOTAP) vesicles for 24 h at 4° C. and thenelectrophoresed on 0.5% agarose. Lipoprotein charge was calculated fromelectrokinetic analyses. Results were as follows:

Lipoprotein Surface Lipid concentration Charge (−mV) Added (mg/ml) VLDLLDL HDL None 0 5.5 2.5 11.8 PI 5 6.3 2.9 12.4 10 6.8 3.3 12.8 50 6.9 3.413.2 100 7.1 3.6 13.4 DOTAP 5 4.6 2.2 11.4 10 4.6 1.9 11.2 50 4.1 1.911.0 100 4.1 1.7 10.9

Discussion of the Examples

Aspects of the present invention have been described in Examples 1-2using both rabbits and humans as experimental models. However, similarresults are expected to occur in other animals. Therefore, the presentinvention contemplates using pharmaceutical compositions comprisingnegatively charged phospholipids to lower the level of cholesterol totreat lipid associated diseases, and to clear endotoxin, bacteria,viruses in any animal or human subject. An animal may include, but isnot limited to, monkey, dog, cat, pig, etc. A “subject”, as the term isused herein, is a human or animal subject, unless the context showsotherwise. The animal results are thought to predict accurately theeffect on a human subject, particularly as the animals used in theanimal studies gave similar results to the human studies in the examplescontaining human studies, and because the animals chosen areconventionally used for studies of lipoproteins, cholesterol and thelike because of the similarity of their reactions to those of humans.

Further, although the examples use PI and PS as a representativeexamples of a negatively charged phospholipid that is capable ofmediating the surface charge of the phospholipid composition, andreducing the level of a lipid-soluble compounds in an animal or subject,it is found that other negatively charged phospholipids also displaysimilar properties to those described. Thus, the present inventioncontemplates the preparation and use of other negatively chargedphospholipids in addition to PI, for example but not limited to PS, PG,and PA, within pharmacological compositions that are capable ofregulating the hepatic clearance of lipoprotein-associated compounds inan animal or subject, or as a food additive.

Injection of PI vesicles into a rabbit caused a dramatic increase innegative surface potential of all classes of lipoprotein (FIG. 1). Thisappears to be directly due to the incorporation of the charged lipidinto the lipoproteins. Incubation of PI with plasma or withultracentrifugally isolated HDL, LDL or VLDL in vitro indicates that alllipoproteins can spontaneously adsorb PI in a manner that appearsphospholipid transfer protein (PLTP) independent, as ultracentrifugedlipoproteins are devoid of this protein.

PI mass measurements show that most of the PI is associated with the HDLparticles and the level of PI increases rapidly, from initially beingbelow detection levels, to up to 30% of the total HDL-PL, after 10 min.After this time, the HDL-PI content slowly decreased to about 5-10% ofthe HDL-PL by 3 h. The clearance of PI from the different lipoproteinsmay have been due to either a lipoprotein-cellular transfer or to aselective hydrolysis by a charged phospholipid specific phospholipase.

When the clearance of [³H]-FC from plasma is monitored, the data showthat after the injection of PT, the tracer is much more rapidly clearedfrom the plasma, than compared to the rabbit injected with theequivalent amount of PC (FIG. 5). This clearance is not the result of anincreased conversion of FC to CE, as very little [³H]-CE is detectable,and LCAT is almost completely inhibited at the early time points.Therefore, these results show that PI stimulates a rapid flux andclearance of cholesterol from the plasma compartment. Several lines ofevidence suggest that this clearance of FC may involve an increasedhepatic uptake of this lipid. Without limitation as to theory,therefore, it is thought that PI may directly enhance interactions withthe HDL receptor, SR-BI, within caveolin enriched domains, and promote aselective clearance of both PC and CE through this receptor.

These examples show that injection of a charged phospholipid, candramatically affect cholesterol transport in vivo. The data indicatesthat changes induced in lipoprotein cholesterol metabolism bynegatively-charged phospholipids are a result of altered interactionsbetween the charged phospholipid-enriched lipoproteins and specific cellsurface microdomains, as well as a change in interactions with theplasma proteins involved in lipoprotein remodelling and metabolism.Almost complete inhibition of the enzyme that produces CE paralleledmajor increases in the amount of this lipid in VLDL particles. Thisindicates that the action of LCAT is not required for a reversecholesterol transport. Cholesterol transport into VLDL may instead begoverned by the charge and structural characteristics of plasmalipoproteins, which may regulate interfacial interactions with specificreceptors and cholesterol storage depots on cell surfaces. Cell cultureexperiments indeed show that PI enhances cholesterol uptake from PIenriched plasma. In transformed hepatocytes (HEPG2 cells), PI-enrichmentof plasma or RDL stimulates cellular cholesterol uptake, while PIenrichment of LDL does not. Without wishing to be limited by theory, itis considered that the enhanced uptake of cholesterol from PI-enrichedHDL is not facilitated by a scavenger receptor-BI mediated pathway butis associated with a novel protein kinase C dependent uptake pathway.Therefore, it is considered that control of lipoprotein charge by theadministration of charged compounds will allow for the manipulation ofFC and CE influx/efflux from cell surfaces and the selective control ofplasma cholesterol metabolism.

According to the invention, it is considered that this charge dependentcontrol of cholesterol transport can be utilized to reduce thecholesterol in the blood stream and treat patients with high cholesterollevels (hypercholesterolemia) by using a negatively chargedphospholipid. To evaluate this potential, the cholesterol loweringpotential of PI was tested in Example 8 in the cholesterol-fed rabbitmodel, which is also a well-accepted model of atherosclerosis. It wasshown that treating the animals for only seven days promotes majorreductions in plasma cholesterol levels by selectively removingcholesterol from the LDL and VLDL particles (FIG. 8), and not from theHDL particles. The results were highly significant and greater inmagnitude to that observed in earlier studies with simvastatin or a CETPinhibitor, that were administered to cholesterol-fed rabbits for a 3month period (Okamoto et al.) The study of Okamoto et al. showed thatcholesterol reducing treatments over a longer term (6 months)significantly reduced the development of atherosclerotic plaque in theaorta of the treated rabbits. Inasmuch as the current invention has muchmore marked short-term effects than the compositions used by Okamoto, itis anticipated that long term treatment of hypercholesterolemic rabbitsor humans with negatively charged phospholipids will prevent theformation of atherosclerotic plaque in susceptible arteries.

The presence of a positively charged phospholipid, on the other hand,slows uptake of lipoprotein constituents. This can be used in the shortterm to slow uptake when needed, as for example when it is desired tokeep a drug associated with a lipoprotein in the bloodstream for alonger than normal period.

Thus, according to the present invention there is provided apharmaceutical composition comprising a synthetic or naturally occurringcharged phospholipid. The pharmaceutical composition containing anegatively charged phospholipid is useful for lowering the level ofcholesterol within a subject when administered in a suitable dosage formcomprising anionic phospholipid as described herein. As outlined above,the administration of a negatively charged phospholipid is associatedwith sterol mobilization into bile and its excretion in faeces.

The present invention has been described with regard to preferredembodiments. However, it will be obvious to persons skilled in the artthat a number of variations and modifications can be made withoutdeparting from the scope of the invention as described herein.

REFERENCES

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1. A method for treating a mammal infected with a pathogen selected froma virus or a bacterium, the method comprising administering to saidmammal a therapeutically effective amount of a negatively chargedphospholipid for achieving removal of either said pathogen or anendotoxin derived therefrom from the bloodstream of said mammal.
 2. Themethod of claim 1, wherein the mammal is a human.
 3. The method of claim1, wherein said isolated negatively charged phospholipid isphosphatidylinositol, phosphatidylserine, phosphatidylglycerol, orphosphatidic acid, or any combination thereof.
 4. The method of claim 1,wherein said isolated negatively charged phospholipid isphosphatidylinositol.
 5. The method of claim 1, wherein said isolatednegatively charged phospholipid is phosphatidylserine.
 6. The method ofclaim 1, wherein said isolated negatively charged phospholipid isphosphatidylglycerol.
 7. The method of claim 1, wherein said isolatednegatively charged phospholipid is phosphatidic acid.
 8. The method ofclaim 1, wherein said isolated negatively charged phospholipid isadministered in a dose of 5 micromole to 100 micromole per kg bodyweight of the mammal.
 9. The method of claim 1, wherein said isolatednegatively charged phospholipid is administered in a dose of 5 micromoleto 20 micromole per kg body weight of the mammal.
 10. The method ofclaim 1, wherein said isolated negatively charged phospholipid isformulated as a food additive.
 11. The method of claim 1, wherein saidisolated negatively charged phospholipid is formulated as a medicament.12. The method of claim 11, wherein the medicament is administeredorally, intranasally, transdermally, or by injection.
 13. The method ofclaim 1, wherein said isolated negatively charged phospholipid is in theform of unilamellar vesicles, multilamellar vesicles, multilamellarsheets, dispersion, micellar solutions, emulsions, or microemulsions.14. The method of claim 1, wherein said isolated negatively chargedphospholipid is in the form of a powder.
 15. The method of claim 1,wherein said pathogen is a virus.
 16. The method of claim 15, whereinsaid virus belongs to the Herpes virus family.
 17. The method of claim16, wherein said virus is cytomegalovirus (CMV).
 18. The method of claim1, wherein said pathogen is a bacterium.
 19. The method of claim 18which achieves removal of said endotoxin from the bloodstream.
 20. Themethod of claim 19, wherein said endotoxin is a lipopolysaccharidecomplex.